Ureaplasma Urealyticum and U. Parvum in Sexually Active Women Attending Public Health Clinics in Brazil

Epidemiol. Infect. (2017), 145, 2341–2351. © Cambridge University Press 2017 doi:10.1017/S0950268817001145

Ureaplasma urealyticum and U. parvum in sexually active women attending public health clinics in Brazil

T. N. LOBÃO1,G.B.CAMPOS1,2,N.N.SELIS2,A.T.AMORIM1,S.G.SOUZA2, S. S. MAFRA2,L.S.PEREIRA2,D.B.DOSSANTOS3,T.B.FIGUEIREDO2, 1,2 1 L. M. MARQUES * AND J. TIMENETSKY 1 Instituto de Ciências Biomédicas, Departamento de Microbiologia, Universidade de São Paulo, Brazil 2 Instituto Multidisciplinar em Saúde, Núcleo de Tecnologia em Saúde, Universidade Federal da Bahia, Brazil 3 Centro de Ciências da Saúde, Universidade Federal do Recôncavo Baiano, Instituto de Ciências Biomédicas, Av. Professor Lineu Prestes, 1374. CEP 05508900, São Paulo, SP, Brazil

Received 15 June 2016; Final revision 5 May 2017; Accepted 16 May 2017; ﬁrst published online 22 June 2017

SUMMARY Ureaplasma urealyticum and U. parvum have been associated with genital infections. The purpose of this study was to detect the presence of ureaplasmas and other sexually transmitted infections in sexually active women from Brazil and relate these data to demographic and sexual health, and cytokines IL-6 and IL-1β. Samples of cervical swab of 302 women were examined at the Family Health Units in Vitória da Conquista. The frequency of detection by conventional PCR was 76·2% for Mollicutes. In qPCR, the frequency found was 16·6% for U. urealyticum and 60·6% U. parvum and the bacterial load of these microorganisms was not signiﬁcantly associated with signs and symptoms of genital infection. The frequency found for Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis and Chlamydia trachomatis was 3·0%, 21·5%, 42·4% and 1·7%, respectively. Higher levels of IL-1β were associated with control women colonized by U. urealyticum and U. parvum. Increased levels of IL-6 were associated with women who exhibited U. parvum. Sexually active women, with more than one sexual partner in the last 3 months, living in a rural area were associated with increased odds of certain U. parvum serovar infection.

Key words: Ureaplasma parvum, Ureaplasma urealyticum, urogenital infection.

INTRODUCTION most of these infections are asymptomatic [2]. These Mollicutes Ureaplasma urealyticum and U. parvum are members of are also responsible for a variety of diseases the class Mollicutes, commonly referred to as mycoplas- and events such as non-gonococcal urethritis, endomet- ritis, pelvic inﬂammatory disease (PID), chorioamnio- mas. Both species are commensals of the human urogenital tract. Infection rates as high as 40–80% in nitis, spontaneous abortion, premature birth, stillbirth women and 50% in men have been reported [1] and and neonatal bronchopulmonary dysplasia [3]. In the early days, U. urealyticum was divided into 14 serovars grouped into two biovars; biovar 1 com- * Author for correspondence: L. M. Marques, Instituto de Ciências posed of serovars 1, 3, 6, 14, while biovar 2 had 10 ser- Biomédicas, Departamento de Microbiologia, Universidade de São ovars 2, 4, 5, and 7–13, respectively. In 2002, biovars 1 Paulo, Brazil; Instituto Multidisciplinar em Saúde, Núcleo de Tecnologia em Saúde, Universidade Federal da Bahia, Brazil and 2 were renamed U. parvum and U. urealyticum, (Email: [email protected]) respectively. This reclassiﬁcation was based on

differences based on genome size, 16S rRNA gene symptomatic women and 64 asymptomatic as a con- sequences, the 16S–23S rRNA intergenic region, trol group. Two women had missing observations enzyme polymorphisms, DNA–DNA hybridization, related to symptoms of genital infection, but kept in and differences in the multiple-banded antigen (mba) the overall prevalence calculations. The following genes [4]. women were excluded: sexually inactive women, The main characteristic of Ureaplasma spp. is their those using antibiotics in the last 3 months, pregnant ability to hydrolyze urea and release ammonia causing and HIV-positive women. The cervix of selected a local cytotoxic effect. In addition they also produce women was accessed using a sterile speculum, and IgA protease, urease, phospholipases A and C, and exudate was removed with a sterile swab. Two swabs hydrogen peroxide [5]. were collected from each woman. One was then placed The opportunistic role of mollicutes includes some in 5 ml of sterile transport medium [12] and stored at features of the innate immune response [6]. Ryckman −20 °C for molecular analysis. The other swab was et al. [7] observed that vaginal IL-1β, IL-6, and IL-8 then placed in a tube containing 1 ml phosphate- levels in pregnant were significantly higher in women buffer saline solution and stored at −70 °C for cyto- with any mycoplasma compared with those without. kines analysis. This may be related to bacterial surface molecules. A questionnaire that surveyed demographic details, Recognition of bacterial surface molecules by the obstetric and gynecologic history and knowledge innate immune system can trigger the production of about sexual behavior was administered. The ques- various proinflammatory cytokines from manifold tionnaire and the study were approved by the ethics cells, a surface component that causes inflammation committee of the Institute of Biomedical Sciences, [8–10]. Therefore it is possible to conclude that the University of São Paulo, Brazil. immune response to mollicutes is variable due the host and the antigenic mosaic and variation of mollicutes. This may explain in part the potential role of DNA extraction Mollicutes in mammals to be excellent opportunistic Swabs were vigorously homogenized in a transport microorganisms that modulate the immune response medium and 1 ml of medium was used for the extraction. differently [11]. The DNA was extracted following the manufacturer’s When these Mollicutes are identified in clinical sam- instructions for Invitrogen Purelink Genomic DNA ples, U. parvum are observed to be more frequent than Kits (Invitrogen, São Paulo, SP, Brazil). U. parvum U. urealyticum. However, this distribution is contro- ATCC 27815 and U. urealyticum ATCC 33175 were versial due the antigenic variation or identification used as standard strains. The standard strains of methods of these Mollicutes. In this context, the aim Ureaplasma were the positive control, and UltraPure™ of the present study was to detect the prevalence of DNase/RNase-Free Distilled Water (Life Technology, U. urealyticum, U. parvum, U. parvum serovars, and Brazil) was the negative control for the PCR and qPCR other microorganisms of genital infections among methodology. The DNA of clinical samples and refer- sexually active women attending health clinics in ence strains were stored at −80 °C. Vitória da Conquista, Brazil.

Conventional PCR and real-time TaqMan PCR METHODS This PCR procedure was used for detecting Mollicutes Endocervical samples (as a screening test), as described by van Kuppeveld This analytical cross-sectional study analyzed clinical et al. [13]. Positive samples for Mollicutes were analyzed samples from Vitória da Conquista city, the third lar- for Ureaplasma species. Primers and probes for U. urea- gest city of Bahia State, in the northeast of Brazil. The lyticum (UUureF/UUureR/ProbeUU) and U. parvum women included in the study attended a Brazilian (UPureF/UPureR/ProbeUP) were used for Real-time public health clinic, known as the ‘Unified Health PCR, as described by Cao et al. [14]. All reactions System’. Cervical swabs were obtained from 302 were performed in duplicate in a StepOnePlus real-time women consecutively attending a health clinic in PCR instrument (Applied Biosystems, Brazil). Vitória da Conquista, Brazil, from May to July 2011 Quantification was performed using an absolute quan- and in January 2012. Women with and without symp- tification technique, based on a predetermined standard toms of genital infection were studied, including 236 curve ranging from 107 to 10 microorganisms/μl. The

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data were acquired during the annealing step and ana- The real-time PCR detected 50 positive samples for lyzed using the StepOne Software 2.1 (Applied U. urealyticum and 183 for U. parvum. Co-infection Biosystems, Life Technologies Corporation). The Ct of both species was observed in 14 samples. U. parvum (threshold cycle) of the genome dilutions was plotted serovars 6 and 3/14 were the most frequent isolates in against the log number of genome copies and used as 64 (40·8%) and 62 (39·5%), respectively, followed by input to create the standard curve. Linear regression serovar 1 in 37 samples (23·6%). There were 27 analyses were then applied to calculate the r2 and samples that were cervical positive for U. parvum; slope values. Assuming 100% efficiency if the DNA however, they were negative for all four known template is doubled in each cycle, the PCR efficiency U. parvum serovars. was calculated as E = 10( − 1/slope) − 1, where E is We observed a prevalence of 42·4% (128/302) of G. PCR efficiency. The positive samples for U. parvum vaginalis infection, 21·5% (65/302) of N. gonorrhoeae, were then submitted to a specific conventional PCR to 3·0% (9/302) of T. vaginalis and 1·7% (5/302) of C. tra- detect serovars 1, 3, 6, and 14 of U. parvum [15]. chomatis. Among women with U. urealyticum, four were coinfected with T. vaginalis, nine with N. gonorrhoeae, 25 with G. vaginalis and one with C. trachoma- Other sexually transmitted microorganism detection tis. However, among women with U. parvum, nine were Cervical samples were also tested with conventional co-infected with T. vaginalis, 40 with N. gonorrhoeae,82 PCR methodology to detect the targeted DNA of with G. vaginalis and four with C. trachomatis. Chlamydia trachomatis [16], Neisseria gonorrhoeae [16], Among the organisms determined by real-time PCR, Trichomonas vaginalis [17]andGardnerella vaginalis [18]. the number of U. urealyticum was 50 (16·6%) of the cervical samples (bacterial load ranging from 10 to 104 organisms per sample). U. parvum organisms were β Measurement of IL-6 and IL-1 detected in 183 (60·6%) of the samples (ranging from The cervical interleukin (IL)-6 and IL-1β levels were dos- 101 to 107 organisms per sample). The bacterial load age in duplicate using a commercially available ELISA of U. urealyticum was significantly higher (P = 0·033, kit (eBioscience, San Diego, CA, USA). To compare Mann–Whitney test) in the asymptomatic group than the difference between IL-6 and IL-1β titers, the values in the symptomatic group. In contrast, there was were log-transformed before statistical analysis. no significant difference between the bacterial of U. parvum in women with and without symptoms. Table 1 shows the demographic, reproductive, and Statistical data analysis behavioral characteristics of the women screened for Statistical analyses of the questionnaire results were U. urealyticum and U. parvum infections detected by conducted through software SPSS 16.0 (SPSS Inc., qPCR. Women living in rural areas (OR 2·85, 95% Chicago, IL, USA). To check association between CI 1·50–5·41, P = 0·001) and having more than one variables, a Pearson a χ2 Test or Fisher’s exact Test sexual partner in the last 3 months (OR 8·07, 95% were used, P < 0·05 and 95% CI. The odds ratio CI 1·08–60·35, P = 0·016) were significantly associated (OR) was computed in Univariate Analysis to esti- with the U. urealyticum infection. However, women mate the power of association between presence of with an active sex life (OR 3·25, 95% CI 1·39–7·57, vaginal infection, with risk factors such as demo- P = 0·004) and having more than one sexual partner graphics and women’s sexual health. A Multivariate in the last 3 months (OR 3·79, 95% CI 1·82–7·88, Analysis Logistic Regression was also used to deter- P < 0·001) were associated with the U. parvum mine which variables were independently associated infection. with infection. In the multivariate analyses, the following factors Mann–Whitney test was used to compare the titers were found to be associated with U. urealyticum infection of cervical IL-6 and IL-1β between individuals with U. (Table 1): living in a rural area (adjusted odds ratio urealyticum or U. parvum infections. (AOR) 2·4, 95% CI 1·2–4·6), with five or more lifetime sexual partners (AOR 3·8, 95% CI 1·1–13·2) and having more than one sexual partner in the last 3 months (AOR RESULTS 5·3, 95% CI 0·6–44·1) had a higher risk, and those not in a The PCR methodology showed that 230 (76·2%) of stable relationship (AOR 0·7, 95% CI 0·2–2·0) had a the women studied were colonized by Mollicutes. lower risk for U. urealyticum infection.

Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.19, on 27 Sep 2021 at 20:25:30, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001145 https://www.cambridge.org/core/terms Downloaded from https://www.cambridge.org/core Table 1. Characteristics of study participants and association with Ureaplasma urealyticum and U. parvum infections in women from Vitória da Coquista, others and Lobão N. T. 2344 Brazil

Ureaplasma urealyticum Ureaplasma parvum (positive) (N = 50) (positive) (N = 183) . https://doi.org/10.1017/S0950268817001145 Variables n (%)* OR ( 95% CI) AOR (95% CI) n (%)* OR ( 95% CI) AOR (95% CI)

Demographic data . IPaddress: Area Rural 21 (42·0) 2·85 (1·50–5·41)a 2·4 (1·2–4·6) 48 (26·2) 1·41 (0·81–2·45) –‡ Urban 29 (58·0) 135 (73·8) 5 170.106.33.19 Reason for visit Symptomatic 17 (34·7) 1·39 (0·72–2·66) –‡ 53 (29·4) 1·07 (0·64–1·80) –‡ Routine 32 (65·3) 127 (70·6) Age , on <25 years 11 (22·0) 1·45 (0·68–3·07) –‡ 34 (18·6) 1·28 (0·69–2·39) –‡

27 Sep2021 at20:25:30 525 years 39 (78·0) 149 (81·4) Race Black/Indigenous 41 (82·0) 1·65 (0·76–3·58) –‡ 140 (76·5) 1·25 (0·74–2·12) –‡ Caucasian/Asian 9 (18·0) 43 (23·5) Education 15 years 35 (70·0) 141 (77·0) Sex life1 Active 49 (98·0) 5·42 (0·72–40·96) –‡ 174 (95·1) 3·25 (1·39–7·57)b 1·8 (0·5–5·9) Inactive 1 (2·0) 9 (4·9) Libido Decreased 19 (38·0) 0·71 (0·38–1·33) –‡ 81 (44·8) 0·99 (0·62–1·58) –‡ Maintained 31 (62·0) 100 (55·2) Lifetime sexual partners 55 7 (14·0) 1·88 (0·75–4·72) –‡ 19 (10·4) 1·59 (0·67–3·77) –‡ <5 43 (86·0) 164 (89·6) Sexual partner in last 3 months§ 51 48 (98·0) 8·07 (1·08–60·35)a 5·3 (0·6–44·1) 169 (93·4) 3·79 (1·82–7·88)a 2·6 (0·9–6·8) None 1 (2·0) 12 (6·6) https://www.cambridge.org/core/terms Downloaded from

Table 1 (cont.) urealyticum Ureaplasma

https://www.cambridge.org/core Ureaplasma urealyticum Ureaplasma parvum (positive) (N = 50) (positive) (N = 183)

Variables n (%)* OR ( 95% CI) AOR (95% CI) n (%)* OR ( 95% CI) AOR (95% CI)

Condom use . https://doi.org/10.1017/S0950268817001145 Rare/occasional 36 (72·0) 0·58 (0·29–1·17) –‡ 138 (78·0) 0·74 (0·40–1·34) –‡ Always 14 (28·0) 39 (22·0)

Hormonal contraception use and

. IPaddress: Yes 30 (60·0) 0·86 (0·48–1·60) –‡ 112 (62·6) 0·96 (0·59–1·55) –‡

No 20 (40·0) 67 (37·4) parvum U. Dyspareunia2 Yes 18 (36·0) 1·28 (0·67–2·42) –‡ 61 (34·1) 1·37 (0·82–2·29) –‡ 170.106.33.19 No 32 (64·0) 118 (65·9) Postcoital bleeding3 Yes 5 (10·0) 1·17 (0·42–3·28) –‡ 16 (8·9) 1·00 (0·44–2·99) –‡ 2345 Brazil in clinics health public attending women active sexually in

, on No 45 (90·0) 164 (91·1) Dysuria 27 Sep2021 at20:25:30 Yes 11 (22·0) 1·06 (0·51–2·21) –‡ 42 (23·0) 1·31 (0·74–2·34) –‡ No 39 (78·0) 141 (77·0) Pelvic pain Yes 24 (48·0) 1·17 (0·64–2·15) –‡ 87 (47·5) 1·34 (0·84–2·14) –‡ No 26 (52·0) 96 (52·5) Itch

, subjectto theCambridgeCore termsofuse,available at Yes 18 (36·0) 1·41 (0·74–2·66) –‡ 61 (33·3) 1·55 (0·92–2·61) 1·5 (0·8–2·6) No 32 (64·0) 122 (66·7) Discharge4 Yes 24 (48·0) 1·18 (0·64–2·17) –‡ 89 (48·9) 1·57 (0·98–2·52) 1·3 [0·7–2·3] No 26 (52·0) 93 (51·1)

AOR, adjusted odds ratio; OR, odds ratio. Missing observations: 1 Sex life: loss of 1 (n = 301). 2 Dyspareunia: loss of 10 and excluded women in inactive sex life (n = 292). 3 Postcoital bleeding: loss of nine and excluded women in inactive sex life (n = 293). 4 Discharge: loss of 1 (n = 301). 5 Reason for visit: loss of 4 (n = 298). * Percentage of total study population with characteristic that tested positive for U. urealyticum and U. parvum by qPCR. § Sexual partners in the last 3 months. ‡ Variables with a p value >0·10 were not included in the multivariate analysis. a P < 0·001. b P < 0·05. 2346 T. N. Lobão and others

Table 2. Multivariate logistic-regression analysis for assessment of independent risk factors for Serovars Ureaplasma parvum infections in women from Vitória da Conquista, Brazil

U. parvum U. parvum

Serovar 1 Serovar 3/14

Independent factor AOR 95% CI AOR 95% CI

Rural area 3·4 (1·6–7·3) –– Education (

* Sexual partners in the last 3 months. AOR, adjusted odds ratio; CI, conﬁdence interval.

Sexually active women (AOR 1·8, 95% CI 0·5–5·9), Significant differences were observed between IL-6 with more than one sexual partner in the last 3 months and IL-1β concentrations in cervical samples among (AOR 2·6, 95% CI 0·9–6·8) and presenting itch (AOR symptomatic and non-symptomatic women (Fig. 1A 1·5, 95% CI 0·8–2·6) and vaginal discharge (AOR 1·3, and B). The association between IL-6 levels did not pre- 95% CI 0·7–2·3) were associated with an increase in sent a significant difference with the U. urealyticum infec- odds for U. parvum infection (Table 1). tions (Fig. 1D). However, higher IL-1β levels were Multivariate analyses of the risk factors associated observed in women with U. urealyticum infection with U. parvum serovar infections are shown in (Fig. 1C). The relationships between cervical IL-1β and Table 2. Women living in rural areas (AOR 3·4, 95% IL-6 levels and U. parvum infection were significant. CI 1·6–7·3), with an education level of less than high Higher IL-1β and IL-6 levels were detected in women school (AOR 1·6, 95% CI 0·6–4·2), dysuria (AOR 1·9, with U. parvum infection (Fig. 1FandG). 95% CI 0·8–4·4) and pelvic pain (AOR 1·3, 95% CI 0·6–2·8) were independently associated with serovar 1 U. parvum. Menarche before 15 years had a lower risk DISCUSSION for infection of serovar 1 U. parvum. Women with a Here we studied a group of women who live in an arid higher number of sexual partners in the last 3 months region of Brazil and compare their age, sexual activity (AOR 4·6, 95% CI 1·1–19·8) and vaginal discharge and relationship status with the molecular detection of (AOR 1·8, 95% CI 0·9–3·4) presented a higher risk to Ureaplasma and other STD agents in cervical swabs. be infected with U. parvum serovars 3/14. Data regarding the relative frequencies of ureaplasmas The reason for the medical care visit (AOR 1·6, in the Brazilian population are scant [19]. Moreover, 95% CI 0·8–2·8), age below 25 years (AOR 2·3, 95% as the separation of human ureaplasmas in two species CI 1·1–4·4), being sexually active (AOR 1·9, 95% CI is recent, the Brazilians studies do not provide this dis- 0·6–6·3), age at first sexual intercourse until 15 years tinction, making it difficult to compare data. In the (AOR 1·6, 95% CI 0·8–2·9), and dyspareunia (AOR present study, U. parvum was detected more fre- 1·5, 95% CI 0·8–2·7) were associated with an increase quently than U. urealyticum. Other studies have also for the odds of G. vaginalis infection (Table 3). shown a higher prevalence of U. parvum in samples A negative association was correlated with living in a of the female urogenital tract than the U. urealyticum rural area (AOR 0·6, 95% CI 0·3–1·1) and an [20–22]. The molecular diagnosis was able to detect education level of less than high school (AOR 0·7, ureaplasmal DNA, but the results do not show viable 95% CI 0·4–1·2). Postcoital bleeding (AOR 3·6, 95% microorganisms as the qPCR results do. But both CI 1·4–9·2) was associated with an increase in the methodologies are fast, and the quantification of odds for N. gonorrhoeae infection, and living in a DNA in a clinical sample is a more accurate indicator rural area (AOR 0·1, 95% CI 0·02–0·3) were associated of infection. In the present study, the bacterial load with decreased odds of N. gonorrhoeae infection. of U. urealyticum was significantly higher in the

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Table 3. Multivariate logistic-regression analysis for assessment of independent risk factors for Gardnerella vaginalis and Neisseria gonorrhoeae infections in women from Vitória da Conquista, Brazil

G. vaginalis N. gonorrhoeae

Independent factor AOR 95% CI AOR 95% CI

Rural area 0·6 (0·3–1·1) 0·1 (0·02–0·3) Reason for visit 1·6 (0·8–2·8) –– Age (<25 years) 2·3 (1·1–4·4) –– Education (

AOR, adjusted odds ratio; CI, conﬁdence interval.

asymptomatic group than in the symptomatic group. neonates [20–22]. However, Jones et al. [28]detected An explanation for this may be related to the commensal inconsistent association of U. parvum infection and characteristic of these microorganisms. Despite the clinical signs. higher bacterial load, other factors related to the host In our study, the prevalence of C. trachomatis was could be related to clinical manifestations [1]. estimated at 1·7%. Yamazaki et al. [29] in Sapporo, In the present study, U. parvum serovars 6 and 3/14 Japan found C. trachomatis in 14·3% of samples. In were the most frequent, followed by serovar Brazil, several studies found this agent ranging from 1. Multiple U. parvum serovars were detected in 18 5·0% to 19·6% among youth attending outpatient cervical samples, while one was not typeable. clinics and gynecological clinics, and attending the Horizontal gene transfer (HGT) has been observed Family Health program [30, 31]. among Ureaplasma species, and the serovars can gen- Three per cent of the studied women were positive erate chimeric isolates with markers of two or more for T. vaginalis. Similarly, a study in the USA found serovars [23]. The HGT was also observed between the prevalence was 3·1% among women aged between U. parvum and Mycoplasma hominis [24]. Xiao et al. 14 and 49 years [32]. Other studies found a higher [23] mentioned the failure to separate multiple sero- prevalence for this microorganism [33, 34]. Here we vars from some clinical isolates, suggesting the occur- found no association between C. trachomatis and T. rence of hybrid isolates and explaining the detection of vaginalis with symptoms of urogenital infection. more than one serovar-specific in PCR assay. Gaydos et al. found an association between Antibody based or PCR methods have reported a cer- Chlamydia and young women, and the number of sex- tain number of non-typeable isolates [25, 26]. ual partners. The T. vaginalis association was found U. urealyticum infection was positively associated with age at first intercourse, previous sexually trans- with the living in a rural area and having more than mitted infections (STIs) and presence of vaginal dis- one sexual partner in the last 3 months, which was charge, and with black women, the number of inversely associated with women not in a stable rela- sexual partners in the year preceding the study, bisex- tionship. Tibaldi et al. [27] found a similar association ual relationships and occasional condom use [35]. with women between 14 and 25 years, without a stable N. gonorrhoeae was detected in 21·5% of samples sexual partner, history of abortion, IUD (intrauterine and was significantly associated with the symptom device) use, more than one sexual partner in the last 6 of postcoital bleeding. Other studies showed lower months and more than two sexual partners during their prevalence of N. gonorrhoeae infection [36, 37]. lifetime. U. parvum infection was more frequently asso- According to Mgone et al. [16] the prevalence of ciated with sexually active women than women with N. gonorrhoeae was highest in young women between more than one sexual partner in the last 3 months 20 and 24 years. G. vaginalis was detected in 42·4% of with itching and vaginal discharge. Other studies have vaginal samples, and was associated with symptom- found an association with this Ureaplasma and PID, atic women, age <25 years, sexually active, age at premature birth and bronchopulmonary dysplasia in first intercourse and symptom dyspareunia. A study

Fig. 1. Relationship between vaginal infection by U. urealyticum, and the concentrations of IL-1β and IL-6 in cervical samples using ELISA among women attending health units in Vitória da Conquista, Brazil, 2013. (A) Concentration of IL-1β (pg/mL) in cervical samples of women and symptomatic and controls. (B) Concentration of IL-6 (pg/mL) in cervical samples of symptomatic women and controls. (C) Concentration of IL-1β (pg/ml) in cervical samples of women qPCR positive and negative U. urealyticum. (D) Concentration of IL-6 (pg/ml) in cervical samples of women qPCR positive and negative U. urealyticum. (E) Concentration of IL-1β (pg/ml) in cervical samples of women qPCR positive and negative U. parvum. (F) Concentration of IL-6 (pg/ml) in cervical samples of women qPCR positive and negative U. parvum. Statistical analysis by Mann–Whitney. P < 0·05.

by Fethers et al. [38] observed an increase in preva- healthcare service for women was not in service for sev- lence of this parasite in women according to their eral months. We believe this factor associated with lack number of sexual partners. of knowledge regarding prevention and treatment of The difference in prevalence between the ureaplas- STIs may have contributed to this prevalence obtained. mas and the searched microorganisms and risk factors Bacterial infections in the vagina typically induce a compared with the literature could be related to the local immune response in the host. The population studied. Some authors confirm that the pro-inflammatory cytokine, IL-1β, is induced in the prevalence of genital microorganisms is related to lower genital tract in women with vaginal infection regional differences [39–44]. However, explanations [45, 46]. The vaginal production of additional cyto- for these differences are not clear, are variable and kines, both pro- and anti-inflammatory, in relation require more study. The women studied live in the to the local immune response to bacterial vaginosis Northeast region of Brazil, which has a semi-arid cli- has not been thoroughly investigated [47]. In this mate, with high rates of poverty. This population study, a statistically significant association was not depends strictly on basic governmental health services. found between both IL-6 and IL-1β and ureaplasma At the time of collection of clinical material, the infection compared with women without infection.

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The proinflammatory cytokine IL-6 is a component do Estado de São Paulo (2011/10138-4). The authors of mechanisms that regulate innate immunity. The thank Aricelma P. França for invaluable technical assist- IL-6 is produced in the early phase of infections, ance, Dr Jorge Sampaio, associated researcher of Fleury and several studies have shown that preterm labor is Institute/São Paulo – Brazil, for kindly providing bacter- associated with increased levels of IL-6 in maternal ial samples and AcademicEnglishSolutions.com for cervicovaginal and amniotic fluid [48]. A difference revising the English. in the IL-6 levels was also detected among women with U. parvum infection. Some studies have shown a positive association between levels of IL-6 in cervi- DECLARATION OF INTEREST covaginal fluid with preterm labor [49] and presence None. of cervical inflammation, altered vaginal microbiota and pregnancy [50]. In the present study, a difference in the IL-6 and REFERENCES IL-1β levels was observed among women with U. urealyticum and U. parvum infection. The IL-1β cyto- 1. Yi J, Yoon BH, Kim EC. Detection and biovar discrimination of Ureaplasma urealyticum by real-time PCR. kines are present intracellularly in the epithelial cells Molecular and Cellular Probes 2005; 19(4): 255–260. at the cell damage. These cytokines play a central 2. Volgmann T, Ohlinger R, Panzig B. Ureaplasma role in regulating the inflammatory response in urealyticum-harmless commensal or underestimated damaged tissue and assist in the development of the enemy of human reproduction? A review. Archives of – preterm labor when associated with an infection, Gynecology and Obstetrics 2005; 273(3): 133 139. 3. Waites KB, et al. Congenital and opportunistic infec- with prostaglandin production by amniotic epithelium tions: ureaplasma species and Mycoplasma hominis. and stimulate myometrial contractily [51]. According Seminars in Fetal & Neonatal Medicine 2009; 14(4): to Patterson et al. [52] isolation of Ureaplasma from 190–199. the respiratory tract of preterm infants is associated 4. Xiao L, et al. Detection and characterization of human with increased IL-1β concentrations. However, Ureaplasma species and serovars by real-time PCR. – another study found no relationship between U. urea- Journal of Clinical Microbiology 2010; 48(8): 2715 2723. lyticum detection and the concentration of any cyto- 5. Sung TJ. Ureaplasma infections in pre-term infants: kine IL-1β, IL-4 and IL-6 in asymptomatic pregnant recent information regarding the role of Ureaplasma women [53]. species as neonatal pathogens. Korean Journal of The high prevalence of microorganisms involved Pediatrics 2010; 53(12): 989–993. et al with STIs underscores the urgent need for comprehen- 6. Doh K, . Differential vaginal expression of interleukin-1 system cytokines in the presence of sive prevention and control programs including not Mycoplasma hominis and Ureaplasma urealyticum in only behavioral interventions but also screening and pregnant women. Infectious Diseases in Obstetrics and improvements in medical care. Sexually active Gynecology 2004; 12(2): 79–85. women, with more than one sexual partner in the 7. Ryckman KK, Williams SM, Kalinka J. Correlations of last 3 months, living in a rural area were associated selected vaginal cytokine levels with pregnancy-related with an increase of odds for ureaplasma infection. traits in women with bacterial vaginosis and mycoplasmas. Journal of Reproductive Immunology 2008; 78(2): This study detected 50 positive samples for U. urealy- 172–180. ticum and 183 for U. parvum and significant differ- 8. Garcia J, et al. A Mycoplasma fermentans-derived syn- ences were observed between IL-6 and IL-1β thetic lipopeptide induces AP-1 and NF-kappaB activ- concentrations in cervical samples among symptom- ity and cytokine secretion in macrophages via the atic and non-symptomatic women. Moreover, IL-1β activation of mitogen-activated protein kinase pathways. The Journal of Biological Chemistry 1998; 273 was observed in higher concentration in women with (51): 34391–34398. U. urealyticum and U. parvum infection. Higher IL-6 9. Kacerovsky M, et al. Amniotic fluid protein profiles of only was observed in women with U. parvum. intraamniotic inflammatory response to Ureaplasma spp. and other bacteria. PloS One 2013; 8(3): e60399. 10. Viscardi RM. Ureaplasma species: role in neonatal mor- ACKNOWLEDGEMENTS bidities and outcomes. Archives of Disease in Childhood Fetal and Neonatal Edition 2014; 99(1): F87–F92. The study has been approved by the Biomedical Science 11. Campos GB, et al. Prevalence of Mycoplasma genitalium Institute Ethics Committee of Sao Paulo University. This and Mycoplasma hominis in urogenital tract of Brazilian study was supported by Fundação de Amparo a Pesquisa women. BMC Infectious Diseases 2015; 15: 60.

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